Functional and physical interactions between partial molecules of STE6, a yeast ATP-binding cassette protein.

The Saccharomyces cerevisiae a-factor transporter, STE6, is a member of the ATP binding cassette (ABC) transporter superfamily. ABC proteins consist of four modular units that comprise two membrane-spanning domains (MSDs) and two nucleotide-binding domains (NBDs). Like many ABC proteins, STE6 contains these four domains in a single polypeptide; certain other ABC proteins are encoded as pairs of "half-molecules" or are further subdivided. Our previous studies demonstrated that STE6 can be expressed as two half-molecules that are functional when co-expressed. Here we dissect the interactions between modules of STE6 in greater detail. We show by co-immunoprecipitation that STE6 half-molecules interact physically, supporting the view that they co-assemble in vivo to form a functional transporter. We also demonstrate a physical interaction between a STE6 half-molecule and full-length STE6; such complexes appear to be functional, based on the striking finding that the defective activity of full-length STE6 mutated in one of its NBDs can be corrected by co-expression of the corresponding "wild-type" half-molecule. We also show that a quarter-molecule consisting solely of the N-terminal MSD of STE6 can interact physically and functionally with a C-terminal three-quarter molecule of STE6, indicating that information directing the assembly of STE6 from partial molecules is contained, at least in part, within its membrane spans.

Comparative topology studies in Saccharomyces cerevisiae and in Escherichia coli. The N-terminal half of the yeast ABC protein Ste6.

Gene fusions have provided a strategy for determining the topology of polytopic membrane proteins in Escherichia coli. To evaluate whether this highly effective approach is applicable to heterologously expressed eukaryotic integral membrane proteins, we have carried out a comparative topological study of the eukaryotic membrane protein Ste6 both in bacteria and in yeast. Ste6, is an ATP binding cassette (ABC) protein, essential for export of the a-factor mating pheromone in Saccharomyces cerevisiae. The topogenic reporters, invertase in S. cerevisiae and alkaline phosphatase in E. coli, were fused to Ste6 at identical sites and the fusions were expressed in yeast and bacteria, respectively. The results obtained in both systems are similar, although more definitive in E. coli, and support the predicted six-transmembrane spans organization of the N-terminal half of Ste6. Thus, the topological determinants for membrane insertion of polytopic proteins in prokaryotic and in eukaryotic systems appear to be highly similar. In this study we also demonstrate that Ste6 does not contain a cleaved signal sequence.

Purification and characterization of sulfide-quinone reductase, a novel enzyme driving anoxygenic photosynthesis in Oscillatoria limnetica.

An enzyme catalyzing sulfide quinone oxido-reduction (E.C.1.8.5.'.; SQR) has been purified in an active form, from thylakoids of the cyanobacterium Oscillatoria limnetica. It is composed of a single polypeptide of about 57 kDa. The catalytic activity of the purified enzyme is similar to the membrane-bound form in its kinetic parameters: apparent Km for sulfide equals 8 microM; Vmax of 100-150 mumol of plastoquinone-1 reduced/mg protein/h; quinone-substrate specificity; differential sensitivity to quinone analog inhibitors, the most potent of which being aurachin C (I50 = 7 nM), and specific inducibility by sulfide. Taken together, they suggest that the purified SQR is the enzyme catalyzing anoxygenic photosynthesis in cyanobacteria. The UV and visible absorption and fluorescence spectra of the purified SQR are typical of a flavoprotein. Both the absorption and fluorescence intensities are reduced by sulfide. The SQR activity is inhibited by KCN, a flavoprotein inhibitor. We have sequenced so far 29 amino acid residues of the SQR NH2 terminus and found that from the second residue, this sequence contains the highly conserved fingerprint of the NAD/FAD-binding domain of many NAD/FAD-binding enzymes (Wierenga, R. K., Terpstra, P., and Hol, W. G. S. (1986) J. Mol. Biol. 187, 101-107). This suggests that the SQR enzyme is a flavoprotein which contains binding sites for sulfide and quinone and that the electron transfer between the two is mediated by FAD.

Mechanism of Na+/H+ exchange by Escherichia coli NhaA in reconstituted proteoliposomes.

Purified NhaA, a Na+/H+ antiporter from Escherichia coli, reconstituted into proteoliposomes was used to study partial reactions catalyzed by this protein. Homologous Na+/Na+ exchange as well as Na+/Li+ exchange via NhaA were detected by monitoring the effects of external Li+ and Na+ ions on the delta pH-driven sodium uptake into NH4 Cl-loaded vesicles. Furthermore, a sodium counterflow reaction was demonstrated in proteoliposomes preloaded with non-radioactive Na+ and placed into the experimental buffer containing low amounts of 22Na+ under experimental conditions when both components of protonmotive force generated by the antiporter. delta psi and delta pH, were dissipated by corresponding ionophores. The apparent Km for sodium counterflow is 1.1 mM, and Vmax is 80 mumol/min/mg of protein. External Na+ accelerates the downhill efflux of 22Na+ suggesting that the translocation of the Na(+)-loaded form of the carrier is faster than the rest of the catalytic cycle.

Proton-sodium stoichiometry of NhaA, an electrogenic antiporter from Escherichia coli.

The H+:Na+ exchange stoichiometry of NhaA, a sodium-proton antiporter coded by the nhaA gene of Escherichia coli, has been determined using purified NhaA protein reconstituted into sodium-loaded proteoliposomes. One approach involved measuring, in parallel experiments, the Na+ efflux and H+ influx from such proteoliposomes and calculating the stoichiometry from the ratio of these fluxes. A second approach was based on measuring the membrane potential generated by NhaA at various sodium gradients and assuming complete coupling and thermodynamic equilibrium between the membrane potential and the ion gradients. The results from both methods agree with a stoichiometry of 2 H+ exchanged for each Na+. This value is independent of pH between pH 7.2 and 8.1. These results support the suggestion that a change in the catalytic rate of NhaA rather than its stoichiometry is crucial for its role in regulation of intracellular pH in alkaline environments.

Histidine-226 is part of the pH sensor of NhaA, a Na+/H+ antiporter in Escherichia coli.

The nhaA gene of Escherichia coli, which encodes a pH-activated Na+/H+ antiporter, has been modified; six of its eight histidine codons were mutated to arginine codons by site-directed mutagenesis, yielding the mutations H254R-H257R (a double mutant), H226R, H39R, H244R, and H319R. In addition a deletion (delta nhaA1-14) lacking the remaining two histidines, His-3 and His-5, has been constructed. By comparing the phenotypes conferred by plasmids bearing the various mutations to the phenotype of the wild type upon transformation of strains NM81 (delta nhaA) or EP432 (delta nhaA, delta nhaB) we found that none of the NhaA histidines are essential for the Na+/H+ antiporter activity of the NhaA protein. However, the replacement of His-226 by Arg markedly changes the pH dependence of the antiporter. All mutants except H226R confer to NM81 and EP432 Na+ resistance up to pH 8.5 as well as Li+ resistance. Cells bearing H226R are resistant to Li+ and to Na+ at neutral pH, but they become sensitive to Na+ above pH 7.5. Analysis of the Na+/H+ antiporter activity of membrane vesicles derived from H226R cells shows that the mutated protein is activated by pH to the same extent as the wild type. However, whereas the activation of the wild-type NhaA occurs between pH 7 and pH 8, that of H226R antiporter occurs between pH 6.5 and pH 7.5. Furthermore, while the wild-type antiporter remains almost fully active at least up to pH 8.5, H226R is reversibly inactivated above pH 7.5, reaching 10-20% of the maximal activity at pH 8.5. We suggest that His-226 is part of a pH-sensitive site that regulates the activity of NhaA.

NhaR, a protein homologous to a family of bacterial regulatory proteins (LysR), regulates nhaA, the sodium proton antiporter gene in Escherichia coli.

On the basis of protein homology, nhaR has previously been shown to belong to a large family of regulatory proteins, the LysR family (Henikoff, S., Haughn, G.W., Calvo, J.M., and Wallace, J.C. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 6602-6606). In this work we show that nhaR is a regulator of nhaA, a gene encoding a Na+/H+ antiporter in Escherichia coli. Multicopy plasmid bearing nhaR enhances the Na(+)-dependent induction of a chromosomal nhaA'-'lacZ fusion. Extracts derived from cells overexpressing nhaR exhibit specific DNA binding capacity to the upstream sequences of nhaA. Construction of an nhaR deletion mutant (OR100) shows that nhaR is required in addition to nhaA to tolerate the extreme conditions under which nhaA is indispensable. Whereas OR100 grows like the wild type at neutral pH even at high Na+ concentrations (700 mM), it becomes much more sensitive to Na+ (greater than 300 mM) at pH 8.5; furthermore, OR100 is more sensitive to Li+ (100 mM) than the wild type. Nevertheless, the phenotype of OR100, which is more resistant to Na+, Li+, and alkaline pH than a delta nhaA strain (NM81), implies that the regulation exerted by nhaR is not complete and that some expression of nhaA exists in OR100. Accordingly, the effect of nhaR in cells is dependent on the level of nhaA. OR200, a nhaA and nhaR deletion mutant, has the same phenotype as NM81. Multicopy plasmid bearing nhaR does not change the phenotype of either OR200 or NM81. On the other hand, multicopy nhaA renders the cells Li(+)- and and Na(+)-resistant even without nhaR.

Overproduction and purification of a functional Na+/H+ antiporter coded by nhaA (ant) from Escherichia coli.

A Na+/H+ antiporter coded by the nhaA (ant) gene of Escherichia coli has been overproduced and purified. The amino-terminal sequence of the protein has been determined and shown to correlate with initiation at a GUG codon, 75 bases upstream from the previously suggested AUG initiation codon. The purified protein, when reconstituted into proteoliposomes, has Na+/H+ antiport activity. It can mediate sodium uptake when a transmembrane pH gradient is applied. Downhill sodium efflux is shown to be highly dependent on pH and is accelerated by a transmembrane pH gradient. An imposed membrane potential negative inside accelerates Na+ efflux at all pH values tested. These findings suggest that the antiporter is electrogenic both at acid and alkaline pH. The activation at alkaline pH values (2000-fold increase) is consistent with the proposed role of the antiporter in regulation of internal pH at the alkaline pH range.

Deletion of ant in Escherichia coli reveals its function in adaptation to high salinity and an alternative Na+/H+ antiporter system(s).

We have deleted the chromosomal ant gene from Escherichia coli by substitution with the kan gene, which encodes kanamycin resistance. The delta ant strains obtained cannot adapt to high sodium concentrations (700 mM, pH 6.8), which do not affect the wild type. The Na+ sensitivity of delta ant is pH dependent, increasing at alkaline pH. Thus at pH 8.5, 100 mM NaCl retard growth of delta ant with no effect on the wild type. The delta ant strains also cannot challenge the toxic effects of Li+ ions, a substrate of the Na+/H+ antiporter system. However, growth of these strains is normal on carbon sources which require Na+ ions for transport and growth. Moreover, antiporter activity, as measured in everted membrane vesicles, is not significantly impaired. A detailed analysis of the remaining antiporter activity in a delta ant strain reveals kinetic properties which differ from those displayed by the ant protein: (a) Km for transport of Li+ ions is about 15 times higher and (b) the activity is practically independent of intracellular pH. Our results demonstrate the presence of an alternative Na+/H+ antiporter(s) in E. coli, additional to ant system.

Sequencing of the gene ant which affects the Na+/H+ antiporter activity in Escherichia coli.

By introduction of deletions and/or insertions, we have defined a small DNA fragment (1.58 kilobase pairs) which contains the ant gene. Thus, transformants of all plasmid constructs containing this segment exhibit Antup phenotype, i.e. growth is Li+-resistant and Na+/H+ antiporter activity is increased in isolated everted membrane vesicles. Utilizing the T7 promoter expression system, we also found that this fragment encodes for a single protein of 35 kDa. The DNA fragment has been sequenced and found to contain an open reading frame of 1085 base pairs. Analysis of the sequence of the predicted protein suggests the presence of 10 putative transmembrane segments in the protein.

An alkaline shift induces the heat shock response in Escherichia coli.

Activation of heat shock response was observed after an alkaline shift of extracellular pH: it peaked at 5 to 10 min, as was previously reported for the heat-induced response, and was dependent on a functional rpoH gene, which is the positive regulator of the heat shock response. An induction of over sixfold was observed for dnaK and groE. The response was induced by the alkalization of extracellular pH but not by the alkalization of intracellular pH. An acidic shift of extracellular pH failed to activate the heat shock response, showing that the response is specific to the alkaline shift.